PROJECT SUMMARY/ABSTRACT Drug resistance represents a major problem in glioblastoma (GBM), the most common and aggressive malignant brain tumor in adults. GBM cells that proliferate and survive through aberrant activation of kinase signaling pathways are believed to avoid the deleterious effects of therapeutic kinase inhibition through utilization of alternative signaling and metabolic pathways. The epidermal growth factor receptor (EGFR) represents a compelling example to dissect this question because at least 40% of human GBMs harbor an EGFR alteration, and inhibitors of this pathway (EGFRi) have been largely ineffective thus far. Despite considerable progress in understanding EGFRi resistance in other cancers (most notably, non-small cell lung cancer), mechanisms of EGFRi resistance in GBM remain poorly understood. To identify novel mechanisms of EGFR kinase inhibitor resistance in GBM, I have performed genome-scale clustered regularly interspaced short palindromic repeats (CRISPR) library screens in several EGFR-mutant GBM cell lines in the presence and absence of the pan-ErbB inhibitor neratinib. Loss of multiple members of the amino-acid sensing pathway (e.g., GCN2, GCN1L1) were associated with neratinib resistance in these cell lines. I have also observed that acute EGFR inhibition induces the amino acid sensing pathway (e.g., phosphorylation of EIF2a and ATF4 expression). Based on these results, I hypothesize that EGFR inhibition induces a state of amino acid starvation and activation of the integrated stress response (ISR) which can be bypassed by loss of GCN2 or other related genes in the ISR pathway. I propose to elucidate this novel mechanism of EGFR inhibitor resistance through a series of biochemical and genetic experiments and further explore its broader biological significance in genetically characterized, patient-derived human GBM models. My overall goal is to further understand the molecular and cellular consequences of EGFR inhibition, with the goal of designing more effective therapeutic strategies for patients with EGFR-altered GBM.